Traditional Chinese Medicine Integrated Department of Nephrology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, P. R. China.
Research Institute of Nephrology, Zhengzhou University, Zhengzhou, P. R. China.
Exp Physiol. 2022 Dec;107(12):1493-1506. doi: 10.1113/EP090685. Epub 2022 Sep 26.
What is the central question of this study? Activation of the glycogen synthase kinase 3 β (GSK-3β)-hypoxia-inducible factor 1 α (HIF-1α) pathway results in stimulation of pyroptosis under high glucose, and exerts actions in a number renal diseases: does this pathway have a role in renal tubular epithelial cells? What is the main finding and its importance? Down-regulation of GSK-3β can inhibit pyroptosis of renal tubular epithelial cells induced by high glucose and this may be related to down-regulation of HIF-1α. This role of the GSK-3β-HIF-1α pathway has not previously been reported and identifies a potential new therapeutic target in diabetic nephropathy.
Diabetic nephropathy (DN) is not only one of the main complications of diabetes, but also has a high incidence rate and a high mortality rate. Glycogen synthase kinase 3 β (GSK-3β) and hypoxia-inducible factor 1 α (HIF-1α) have been demonstrated to influence DN by regulating pyroptosis. This study aimed to investigate the effect of the GSK-3β-HIF-1α pathway on pyroptosis of high-glucose (HG)-induced renal tubular cells. Mouse renal proximal tubular epithelial cells (TKPT cells) were induced by HG to simulate DN cell and we transfected TKPT cells with GSK-3β knockdown lentivirus. Western blot analysis confirmed the transfection effects and detected the expression of GSK-3β, HIF-1α, Nod-like receptor protein 3 (NLRP3), cleaved-caspase-1, pro-caspase-1, gasdermin D (GSDMD) and GSDMD-N. The expression of GSDMD-N and HIF-1α were also verified by immunofluorescence. The levels of interleukin (IL)-1β and IL-18 were measured by enzyme linked immunosorbent assay. Flow cytometric analysis determined the apoptosis rate. Results showed that HIF-1α expression was increased in HG-induced TKPT cells, and GSK-3β knockdown could decrease the levels of NLRP3, cleaved-caspase-1, GSDMD-N and HIF-1α, verified by immunofluorescence. Moreover, GSK-3β knockdown suppressed the expression of IL-1β and IL-18, and reduced the apoptosis rate. Lithium chloride (LiCl) interference could cause the same changes as GSK-3β knockdown for HG-induced TKPT cells, and dimethyloxallyl glycine could reverse the effect of GSK-3β-knockdown interference. Our studies definitively demonstrate that the GSK-3β-HIF-1α signalling pathway mediates HG-stimulated pyroptosis in renal tubular epithelial cells and that down-regulation of GSK-3β inhibited HG-induced pyroptosis by inhibiting the expression of HIF-1α. These findings suggest a new potential target for the treatment of DN.
本研究的核心问题是什么？糖原合酶激酶 3β（GSK-3β）-低氧诱导因子 1α（HIF-1α）通路的激活导致高糖诱导的细胞焦亡，在许多肾脏疾病中发挥作用：该通路在肾小管上皮细胞中是否有作用？主要发现及其重要性是什么？下调 GSK-3β可抑制高糖诱导的肾小管上皮细胞焦亡，这可能与 HIF-1α的下调有关。GSK-3β-HIF-1α通路的这种作用以前没有报道过，为糖尿病肾病提供了一个潜在的新治疗靶点。
糖尿病肾病（DN）不仅是糖尿病的主要并发症之一，而且发病率和死亡率都很高。糖原合酶激酶 3β（GSK-3β）和低氧诱导因子 1α（HIF-1α）已被证明通过调节细胞焦亡来影响 DN。本研究旨在探讨 GSK-3β-HIF-1α通路对高糖（HG）诱导的肾小管细胞焦亡的影响。用 HG 诱导小鼠肾近端小管上皮细胞（TKPT 细胞）模拟 DN 细胞，并转染 GSK-3β 敲低慢病毒。Western blot 分析证实了转染效果，并检测了 GSK-3β、HIF-1α、Nod 样受体蛋白 3（NLRP3）、半胱天冬酶-1 剪切体（cleaved-caspase-1）、前半胱天冬酶-1（pro-caspase-1）、Gasdermin D（GSDMD）和 GSDMD-N 的表达。免疫荧光验证了 GSDMD-N 和 HIF-1α的表达。酶联免疫吸附试验（ELISA）检测白细胞介素（IL）-1β和 IL-18 的水平。流式细胞术分析测定细胞凋亡率。结果表明，HG 诱导的 TKPT 细胞中 HIF-1α表达增加，GSK-3β 敲低可降低 NLRP3、cleaved-caspase-1、GSDMD-N 和 HIF-1α的水平，免疫荧光验证。此外，GSK-3β 敲低抑制了 IL-1β和 IL-18 的表达，并降低了细胞凋亡率。氯化锂（LiCl）干扰可引起与 GSK-3β 敲低对 HG 诱导的 TKPT 细胞相同的变化，二羟乙氧乙氧乙酸（dimethyloxallyl glycine）可逆转 GSK-3β 敲低干扰的作用。我们的研究明确证实，GSK-3β-HIF-1α信号通路介导了肾小管上皮细胞中 HG 刺激的细胞焦亡，下调 GSK-3β 通过抑制 HIF-1α 的表达抑制了 HG 诱导的细胞焦亡。这些发现为糖尿病肾病的治疗提供了一个新的潜在靶点。
